The long term goal of this project is to develop a 384-well plate-based cell migration assay suitable for high throughput screening (HTS) of chemical libraries. The principal barrier to performing HTS to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. The further advancement of OrisTM technology, as described in this proposal, will form the basis of an affordable, easy to use cell-based assay capable of providing relevant data for quickly screening drug candidates for their ability to impact cell migration. The availability of a HTS 384 well cell-based assay that requires minimal numbers of cells and minute volumes of test compounds will facilitate primary screens and better drug development for cancer therapeutics. The proposed HTS assay format will be compatible with automated liquid handling systems and high content screening (HCS) platforms. It will be amenable to primary screens which can be read quickly by plate reader instrumentation. It will also permit subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. This HTS assay is based on the innovative use of a chemically derived cell exclusion zone that will be deposited in a defined central area at the bottom of a tissue culture well. Cells are then seeded and attach at the perimeter of the well, the exclusion zone dissolves and reveals a zone that is now permissible for cell migration. The first generation OrisTM Cell Migration Assay provides 96 wells for investigating the effects of cell movement modulators. It uses silicone stoppers to create exclusion zones. Using the Oris" assay, we demonstrated measurable migration of A-549 cells and reported z-factors of >0.46 for migration to support claims of assay robustness. We have shown that the assay is suitable for testing modulators of cell motility. We further showed the ability to collect multiple pieces of information from a single test well (i.e., high content screening capable) and data analysis compatible with fluorescence microplate readers and imaging platforms. Finally, we provided recent data to support the creation of a reversible polymeric exclusion zone that eliminates the need for a silicone stopper and makes the assay highly amenable for use with automated liquid handlers employed by HTS laboratories. It appears that the polymer completely dissolves as evidenced by the full migration of cells into the previously restricted area and has no obvious deleterious effects on cell viability or test compounds. These data strongly support the feasibility of modifying the Oris" 96-well cell migration assay into a 384-well, high throughput cell migration assay. In this phase I assay, we propose to first develop a 96-well cell migration assay to allow for greater amounts of both primary and secondary data to be obtained from a single assay well by using multiplexed staining techniques with different fluorophor conjugates. The assay will enjoy a wide range of compatibility with a variety of HCS platforms as well as standard fluorescent plate readers and microscopes. Our intended product, a 384 well assay, will streamline the drug discovery process to facilitate quicker screening of molecular libraries for development of therapeutics that block cancer cell metastasis or promote wound healing. PUBLIC HEALTH RELEVANCE: The principal barrier to performing high throughput screening (HTS) to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. Our proposed HTS assay format will be compatible with automated liquid handling systems and high content screening platforms. This assay format will be amenable to primary screens which can be read quickly by plate reader instrumentation while permitting subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. Based on proven success in launching the Oris" product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The goal of this Phase I proposal, is to 1) show feasibility for creating consistently sized and placed polymer deposits in multiwell plates to create chemically derived cell exclusion zones and 2) ability of cells to proliferate and migrate in the presence of motility modulating compounds on these plates.